This Phase I SBIR application examines global changes in human osteoblast protein expression in responsive to osteogenic growth factors. The long-term goals of this proposal are to develop a proteomics approach to human osteoblast function that allows the identification of therapeutic targets for drug discovers. By focusing on primary human osteoblast, such targets are physiologically relevant, and ultimately important to our understanding of bone diseases such as osteoporosis. It is hypothesized that that osteogenic growth factor stimulation results in changes in protein expression/modification that are amenable to proteomic analysis. We further postulate that combinations of known bone-active growth factors can be used as a proof-of-concept approach to identify protein targets. The overall goal of this project is to discover potential cellular targets for stimulating human osteoblast development; its long-term objective is the identification of protein targets that are of therapeutic value in the treatment of bone diseases. This phase I SBIR focuses on demonstrating the feasibility of using proteomics as a discovery tool in human osteoblast development, on demonstrating that cellular fractionation (into membrane, cytosolic, and nuclear proteins) reduces the complexity of the proteomic analysis, and on showing that differentially expressed proteins can be readily, and rapidly identified by combined 2-dimensional gel electrophoresis (2-DGE) and mass spectrometry identification. The proposed work represents a collaboration between Velcura Therapeutics, Inc., who will perform the biological and 2DGE components of the proposal, and Proteomic Research Services, Inc. who will do the mass spectrometry/ protein identification. This proposal represents an innovative blend of human osteoblast cell biology, proteomic analyses, and state-of-the-art mass spectrometry to identify proteins differentially regulated by two synergistically interacting osteogenic growth factors, TGF-beta & BMP2. Uniquely, the cell biology uses primary human osteoblasts grown in serum-free media as 3-dimensional tissue-like aggregates. Thus, the human osteoblasts develop in a more physiological context. It is predicted that the joint Velcura/PRS investigations will result in several distinct outcomes: 1) Establish the protocol, format, and initial population of a protein map database for developing human osteoblasts. 2) Provide a proof-of-concept that 2-DGE Averaged Maps (among and between individual human osteoblasts cell donors) can identify "activation states" of human osteoblasts. 3) Produce a proof-of-concept, using TGFbeta1 and BMP-2, that the protein targets of these molecules can be identified by 2-DGE and MS/MS or LC/MS/MS. 4) Identify between 75 and 525 (depending on the number of proteins resolve by MS in each protein -spot) proteins that are consistently modulated in the membranes, cytosol, and nuclear cell fractions.